Phage therapy to inactivate multidrug-resistant P. aeruginosa

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Universidade de Aveiro 2011

Departamento de Biologia

Anabela Carvalho

Vieira

Phage therapy to inactivate multidrug-resistant

P. aeruginosa

Terapia fágica para inactivar P. aeruginosa

multi-resistente

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Universidade de Aveiro 2011

Departamento de Biologia

Anabela Carvalho

Vieira

Phage therapy to inactivate multidrug-resistant

P. aeruginosa

Terapia fágica para inactivar P. aeruginosa

multi-resistente

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Microbiologia, realizada sob a orientação científica da Professora Doutora Maria Adelaide de Pinho Almeida, Professora Auxiliar do Departamento de Biologia da Universidade de Aveiro.

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o júri Presidente do Júri

Prof. Doutora Maria Ângela Sousa Dias Alves Cunha

Professora Auxiliar

Departamento de Biologia da Universidade de Aveiro

Vogais

Prof. Doutora Maria Adelaide Pinho de Almeida (orientadora) Professora Auxiliar

Departamento de Biologia da Universidade de Aveiro

Prof. Doutora Joana Cecília Valente Rodrigues Azeredo (arguente) Professora Associada

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agradecimentos À Professora Doutora Adelaide Almeida, orientadora da tese, pelo incentivo, confiança, dedicação, paciência e constante disponibilidade.

À Professora Doutora Ângela Cunha pelo sentido crítico e pela simpatia ao longo do trabalho.

Aos técnicos Srª Helena e Srº Armando por todo o apoio técnico e constante disponibilidade.

À Yolanda pela preciosa ajuda e por tudo que me ensinou durante a realização desta dissertação.

Aos meus colegas do Laboratório Ambiental a Aplicada, Joana Almeida, Joana Brás, Adriana, Clara, Patrícia, Ana Luísa, Lia, Eliana, Inês e Vanessa, pela constante disponibilidade e momentos de boa disposição.

A todos os outros colegas, pela boa disposição e apoio.

Aos meus pais, avós e irmão pela força dada, pela paciência demonstrada, pelo amor e pelo apoio incondicional.

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keywords

abstract

Phage therapy, bacteriophage, Pseudomonas aeruginosa, multidrug resistant bacteria, human skin, wound infections

With the increase in antibiotic resistance and after several years of abandonment, the use of bacteriophages (phages), as antimicrobial agents, to destroy bacteria began to arouse interest in the scientific community. This has led to a huge phage research in different fields and currently several studies are ongoing with animals and humans. Pseudomonas aeruginosa is an opportunistic pathogen, which frequently colonizes wounds infections. It has been estimated that a high number of deaths caused by wound infections results of bacterial infection, often by antibiotic-resistant P. aeruginosa. The main target of this work was to explore the potential of phages in controlling multidrug-resistant (MDR) P. aeruginosa strains in vitro and ex vivo (human skin). A new bacteriophages (PA709) was isolated from sewage water samples collected from Hospital Universitário de Coimbra (HUC). A phage suspension (108 PFU mL-1) was obtained using the clinical strain P. aeruginosa 709 as host. After the characterization of the phage candidate, their capacity to lyse other MDR P. aeruginosa clinical isolates from Aveiro, Matosinhos and Coimbra was investigated. The ability of the phage to cause inactivation of P. aeruginosa 709 was evaluated in vitro and in ex vivo (human skin), at 37°C, using a multiplicity of infection (MOI) of 0.5 to 50. In the in vitro assays, the effect of a second dose application, added after 4 hours of incubation, was also tested.

The lytic phage PA709 has an icosahedral head with a long contractile tail and a DNA molecule as nucleic acid, a morphology characteristic of members of the Myoviridae family. The phage PA709 show a relatively broad host range (infects 30% of the 51 MDR P. aeruginosa clinical isolates), infecting different genotypes isolated in the three hospitals (Matosinhos, Aveiro and Coimbra). For the best MOI, the number of MDR P. aeruginosa 709 in the human skin in the presence of the phage decreased 4 logs after 2 hours of incubation. The application of a second dose of phage did not increase the efficiency of the therapy. These results show that the phage PA709 was seen to have rapid lytic activity but the number of bacteria gradually increased after that. The occurrence of lysogeny and the development of resistance of the host to the phages could explain the bacterial re-growth. However, no evidence of lysogeny was observed after addition of mitomycin C and no resistant to PA709 phage was detected.

In conclusion, phage PA709 presents some interesting features, namely high efficiency in the inactivation of MDR P. aeruginosa , a broad host range and high stability in stock suspensions and in ex vivo human skin. All these attributes make this phage very promising for the treatment of P. aeruginosa skin wound infections. However, more phages should be isolated in the future, for the formulation of cocktails which might improve the inactivation efficiency against P. aeruginosa human skin infections.

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Palavras-chave

resumo

Terapia fágica, bacteriófagos, Pseudomonas aeruginosa, bactérias multi- resistentes, pele humana, infecções da pele

Com o aumento da resistência aos antibióticos e após vários anos de abandono, o uso de bacteriófagos (fagos), como agentes antimicrobianos, para destruir bactérias começou a despertar interesse na comunidade científica. Isto levou a uma enorme investigação dos fagos em diferentes áreas e actualmente muitos estudos estão em curso usando animais e humanos. Pseudomonas aeruginosa é um patogénico oportunista, que frequentemente coloniza infecções da pele. Foi estimado que o elevado número de mortes causado por infecções da pele resulta de infecções bacterianas, muitas vezes por P. aeruginosa com resistência aos antibióticos.

O principal objectivo deste trabalho foi explorar o potencial do fago em controlar estirpes de P. aeruginosa multi-resistentes (MR) in vitro e ex vivo (pele humana). Um novo bacteriófago (PA709) foi isolado da água do esgoto do Hospital Universitário de Coimbra (HUC). A suspensão fágica (108 UFP mL-1) foi obtida usando a estirpe clínica P. aeruginosa 709 como hospedeiro. Após a caracterização do fago candidato, a sua capacidade em lisar outros isolados clínicos MR de P. aeruginosa de Aveiro, Matosinhos e Coimbra foi investigada. A capacidade do fago causar inactivação da P. aeruginosa 709 foi avaliada in vitro e in ex vivo (pele humana), a 37ºC, usando uma multiplicidade de infecção (MOI) de 0,5 a 50. Em ensaios in vitro, o efeito da aplicação de uma segunda dose, adicionada após 4 horas de incubação, foi também testada. O fago lítico PA709 tem uma cabeça icosaédrica com uma cauda longa e contráctil e molécula de DNA como ácido nucleico; morfologia característica dos membros da família Myoviridae. O fago PA709 infecta 30% dos 51 isolados clínicos MR de P. aeruginosa, indicando uma infecção relativamente ampla de hospedeiros. Para a melhor MOI, o número de P. aeruginosa 709 MR na pele humana, na presença de fago, diminuiu 4 logs após 2 horas de incubação. A aplicação de uma segunda dose do fago não aumentou a eficiência da terapia. Estes resultados confirmam que o fago PA709 parece ter uma rápida actividade lítica, mas o número de bactérias aumentou gradualmente depois disso. A ocorrência de lisogenia e o desenvolvimento de resistência do hospedeiro ao fago pode explicar o re-crescimento bacteriano. No entanto, não foi observada a presença de lisogenia após a adição de mitomicina C nem a resistência ao fago PA709 foi detectada.

Em conclusão, o fago PA709 apresenta algumas características interessantes, nomeadamente elevada eficiência em inactivar P. aeruginosa MR, uma infecção ampla de hospedeiros e elevada estabilidade na suspensão em stock e na pele humana. Todas estas características fazem este fago muito promissor para o tratamento de infecções na pele de P. aeruginosa. No entanto, no futuro mais fagos deverão ser isolados, para obter cocktails de fagos que podem melhorar eficientemente a inactivação contra infecções na pele humana de P. aeruginosa.

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1. Introduction

1.1. Bacteriophages

1.1.1. Discovery of bacteriophages

The story of the discovery of bacteriophages or phages has been controversial and subject to many debates. In 1896, in India, Ernest Hankin observed in waters of two rivers the existence of high antibacterial activity against Vibrio cholera (Deresinski, 2009). He suggested that an unidentified substance was responsible for this phenomenon. Two years later, identical observation was made by Gameleya, while he worked with Bacillus subtilis (Sulakvelidze et al., 2001). These findings have not been explored and, only 20 years later, this topic has again been introduced (Sulakvelidze et al., 2001).

At the beginning of the twentieth century, Frederick Twort and Felix d'Herelle, independently, described entities that could destroy cultures of bacteria. D'Herelle named them bacteriophages. The name was formed from “bacteria” and “phagein” (to eat or devour, in Greek) (Sulakvelidze et al., 2001). In 1917, d'Hérelle published these observations, describing the general procedures for isolation bacterial viruses. The bacteriologist isolated phages for some pathogenic bacteria that caused diseases like cholera (Skurnik and Strauch, 2006). Moreover, d'Hérelle developed the method of quantification of viruses and other theories, including the replication cycle of the phage (Bratbak and Heldal, 1993).

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1.1.2. Properties and classification of bacteriophages

Bacteriophages are viruses that infect bacterial cells. It has been estimated that phages are ten times more numerous in the environment than bacteria, making them the most abundant entities on Earth (Ackermann, 2007; Skurnik and Strauch, 2006).

Phages have two essential components, proteins and nucleic acids. Bacteriophage taxonomy is based on their shape, size, proteins as well as on their nucleic acid. Most phages have dsDNA, however, some have ssDNA, dsRNA or ssRNA (Matsuzaki et al., 2005). In total there are 17 families of phages (Figure 1.1) (Ackermann, 2001; Ackermann, 2007; Hanlon, 2007).

Figure 1.1: Schematic representation of the families described in the classification of bacteriophages (Ackermann, 2007).

Tailed phages are classified into three families and represent about 96% of the phages reported (Skurnik et al., 2007). These phages are composed of an icosahedral head and tail, and all of them have dsDNA (Table 1.1) (Ackermann, 2001; Ackermann, 2007). The Myoviridae family has got a contractile tail, the Siphoviridae family a long tail not

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contractile and the Podoviridae family a very short tail. These three families comprise the order Caudovirales (Table 1.1) (Ackermann, 2001; Ackermann, 2007; Hanlon, 2007).

The other families, which only constitute 4% of reported phages, are cubic (polyhedral), ﬁlamentous or pleomorphic. They contain ds or ssDNA or RNA as the genome (Table 1.1) (Ackermann, 2001; Ackermann, 2007; Dabrowska et al., 2005).

Table 1.1: Main characteristics of bacteriophages and their classification (Ackermann, 2007).

Order Family Shape Nucleid acid Morphology

Caudovirales Myoviridae Tailed ds DNA, linear Tail contractile

Siphoviridae Tail long, non contractile

Podoviridae Tail short

Microviridae Cubic (polyhedral)

ss DNA, circular Capsomers

Corticoridae ds DNA, circular superhelical Complex capsid, lipids

Tectiviridae ds DNA, linear Inner lipid vesicle, pseudotail

Leviviridae ss RNA, linear Poliovirus-like

Cystoviridae ds RNA, linear segmented Envelope, lipids

Inoviridae Filamentous ss DNA, circular Long ﬁlaments, short rods

Lipothrixviridae ds DNA, linear Envelope, lipids

Rudiviridae ds DNA, linear TMV-like

Plasmaviridae Pleomorphic ds DNA, circular superhelical Envelope, lipids, no capsid

Fuselloviridae ds DNA, circular superhelical Lemon-shaped

Salterprovirus ds DNA, linear superhelical Lemon-shaped

Guttaviridae ds DNA, circular superhelical Droplet-shaped

1.1.3. Bacteriophage infection

The phages are metabolically inert in their extra cellular form. They are only able to self-reproduce as long as the host bacteria is present and their replication depends exclusively on the host intracellular machinery to translate their own genetic code (Dabrowska et al., 2005; Lorch, 1999).

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Viruses can interact with their hosts in two major and distinctive ways, the lytic and lysogenic cycles of infection and more sporadically through pseudolysogeny. However, only lytic phages are suitable candidates for phage therapy since they may destroy bacteria (Almeida et al., 2009; Hanlon, 2007; Weinbauer, 2004).

In the lytic cycle, they multiply in the host cell and lyse the bacterial cell to release newly formed phage particles. Firstly, the phage binds to specific receptors of bacteria (Goodridge, 2010; Weinbauer, 2004). This phase is called adsorption. Phages can use different parts of lipopolysaccharide (LPS), ﬂagella, ﬁmbriae and many other surface proteins as receptors. Bacteriophages may also use enzymes to break down the bacterial surface (Skurnik and Strauch, 2006; Wróblewska, 2006). Then the phage genome is injected into the host bacterium and occurs early gene expression. Most of the proteins produced in this phase are involved in the shutting down of the host bacterium systems and phage genome replication. In some cases, the early proteins degrade the host DNA (Goodridge, 2010; Weinbauer, 2004). After replication of the phage genome, occurs the expression of the phage late proteins that are involved in the formation of new phage particles and lysis of host bacteria (Duckworth and Gulig, 2002). The phage head and tail are assembled and the phage genome is packaged. The bacteria are destroyed through lysis, resulting in an average release of 50 to 200 daughter particles(Huff et al., 2005) (Figure 1.2).

In lisogenic cycle, the phage genome is integrated into the host cell DNA. Prophage DNA will be replicated when the host cell genome replicates and so daughter cells will inherit the viral DNA (Figure 1.2). The prophage can stay in a dormant state for long periods of time and may become activated and turn on the lytic cycle. The lytic cycle is induced spontaneously by chemical or physical agents such as radiation, pollutantes, changes in temperature and nutrient concentrations (Almeida et al., 2009; Weinbauer, 2004). At the end the newly formed phage particles will lyse the host cell. Lysogeny might be a viral survival strategy to ensure periods of low host density during nutrient starvation (Weinbauer, 2004).

There is another phenomenon known as pseudolysogeny. However, unlike true lysogeny, the phage genome does not integrate into the host. Pseudolysogeny is a

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condition in which the starved bacterial cell coexists in an unstable relationship with infectingviruses (Figure 1.2). In such host cells, there is insufficient energy available for the phage to initiate genetic expression leading to either a true temperate response or to the lytic response (Ripp and Miller, 1997). As nutrients are supplied to the bacterium, the pseudolysogens resolve into either true lysogeny or active production of virions (lytic cycle). The direct result of pseudolysogenic relationships is an extension of the effective phage half-lives in naturalenvironments (Almeida et al., 2009; Ripp and Miller, 1997). The pseudolysogenic state wasfound to depend on the concentration of nutrients availableto the host. As cells became more starved, the frequency of pseudolysogens increased (Ripp and Miller, 1997; Weinbauer, 2004).

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1.2. Human skin flora and wound infection

Human skin has intrinsic properties that are important to prevent infection and promoting healing in wounds (Church et al., 2006; Cunha, 1998). This organ provides sensation, thermoregulation, biochemical, metabolic, immune functions and physical protection and prevents infection caused by pathogenic microorganisms (Church et al., 2006).

The normal microflora of the skin includes fungi and bacteria. In 1938, Price reported that microorganisms found on the skin can be divided into resident flora, composed of commensals that rarely damage the host, or transient flora which do not grow on skin and reflects the host level of personal hygiene, lifestyle, personal activities and level of environmental contamination (Price, 1938).

The predominant bacterial resident flora of the skin is various species of coagulase-negative staphylococci (Staphylococcus epidermidis), Corynebacterium spp. and Propionibacterium spp. (Cunha, 1998). The Gram-negative bacteria often colonize healthy adult skin include Proteus sp., Enterobacter sp. and Klebsiella sp., Acinetobacter spp. and Pseudomonas spp., constituting about 25% of the adult skin microflora (Percival et al., 2010).

The bacteria become pathogenic soon they can adhere, grow and invade the host. Typically, soft tissue infections result from disruption of the skin by exogenous factor, extension from subjacent infection or disseminated through the blood stream from a distant site of infection. Most of skin and soft tissue infections are superficial, treated with local care and antimicrobial therapy (Cunha, 1998). Other factors predisposing to skin infections include vascular insufficiency, disrupted venous or lymphatic drainage, diabetes mellitus, previous cellulitis, foreign bodies, accidental or surgical trauma, burns, poor hygiene, obesity and immunodeficiencies (Cunha, 1998).

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Pathogens causing initial infections are usually bacterial and the subsequent infections are caused usually by antibiotic-resistant bacteria. Antibiotics alter the balance of natural flora, leaving the surface vulnerable to colonization by exogenous gram-negative bacilli, yeasts and fungi which, usually occurs later due to the use of broad-spectrum antibiotic therapy (Church et al., 2006).

Colonization with organisms, such as Gram-negative bacilli, is not favored. Enzymes and other metabolic products produced by Gram negative bacteria, enhance the invasive potential and the rapid spread of these infections (Church et al., 2006). Moreover, many Gram negative organisms are resistant to antibiotics, which mean it becomes difficult to eradicate (Tredget et al., 2004). Some bacteria are often organized in biofilms. These bioflms can form within 10 - 72 hours and acts as an effective barrier against host defenses and antimicrobial agents. (Kutter et al., 2010; Rode, 2010). In addition, the immunosuppressive state of the patient and the immediate lack of antibodies, allow multiplication of potential pathogens in the wound (Edwards-Jones and Greenwood, 2003).

Infections by Pseudomonas aeruginosa

P. aeruginosa is a non-fermentative, Gram negative bacilli and oxidase-positive. These bacteria is the main pathogen to cause wound infections, remain a major cause of sepsis, morbidity and high mortality (Church et al., 2006). Cause other diseases such as, pneumonia, bacteremia, meningitis, urinary tract infection, skin and soft tissue infections in immunocompromised individuals and hospitalized patients (Wróblewska, 2006). Colonization is more common in the respiratory tract, gastrointestinal tract and skin (Church et al., 2006). It is an opportunist pathogen that is notoriously unresponsive to many antibiotics. P. aeruginosa have many virulence factors, including structural components, toxins and enzymes (Table 1.2).

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Table 1.2: Virulence factors of P. aeruginosa and its biological effects.

Virulence factors Biological efects Reference

Capsule growth as a biofilm; protection

from innate and immune defenses

(Drenkard and Ausubel, 2002; Govan and Deretic, 1996) Pili

adherence to host (Govan and Deretic, 1996)

Adhesins Lipid A

Toxicity (Govan and Deretic, 1996)

Lipopolysaccharide

Exotoxins inhibition of protein synthesis (Edwards-Jones and Greenwood,

2003; Govan and Deretic, 1996) Elastase

tissue damage (Edwards-Jones and Greenwood,

2003; Govan and Deretic, 1996) Protease

Phospholipase C

The capsule is composed by mucoid polysaccharides, which is important for growth as a biofilm in which bacterial cells are protected from innate and immune defenses, and become less susceptible to antimicrobials (Drenkard and Ausubel, 2002; Govan and Deretic, 1996). Its ability to form biofilm has been suggested to cause failure to heal in chronic wounds. The adherence to host is mediated by pili and adhesins (Govan and Deretic, 1996). The presence of lipid A and lipopolysaccharide (LPS) which is a component of the cell wall, enhances the toxicity of this microorganism (Govan and Deretic, 1996). Various toxins and enzymes are secreted, which causes inhibition of protein synthesis and cell death in the host. This causes local necrosis and can cause septicaemia (Edwards-Jones and Greenwood, 2003; Govan and Deretic, 1996).

1.3 Bacterial resistance to antibiotics

Chemotherapy has shown to be a rapid and effective method to treat or prevent microbial infections, but the regular use of antimicrobials has resulted in the development of drug resistance in common pathogenic microbial strains (Towner and Bergogne-Berezin, 1996). Even though novel classes of antibiotics may be developed, the prospect that

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bacteria will eventually develop resistance to the new drugs (Lorch, 1999), emphasize that effective antibiotics may not be available to treat seriously ill patients in the near future.

Most antimicrobial agents used are categorized according to their principal mechanism of action. There are five major modes of action, disruption of bacterial membrane structure, interference with cell wall synthesis, inhibition of protein synthesis, interference with nucleic acid synthesis and inhibition of a metabolic pathway (Table 1.3) (Tenover, 2006).

Table 1.3: Mechanisms of action of antibacterial agents. Adapted from Tenover (2006).

Mechanisms of action Antibacterial agents

Disruption of bacterial membrane structure Increase bacterial membrane permeability or membrane depolarization

polymyxins, daptomycin

Interference with cell wall synthesis Inhibit synthesis of the bacterial cell wall by interfering with the synthesis of the peptidoglycan layer

β-Lactams: penicillins, cephalosporins, carbapenems, monobactams Glycopeptides: vancomycin, teicoplanin

Protein synthesis inhibition

Inhibit bacterial growth by binding to the 30S or 50S subunit of the ribosome

Macrolides, aminoglycosides, tetracyclines, chloramphenicol, streptogramins, and

oxazolidinones Interference with nucleic acid synthesis

Inhibit DNA or RNA synthesis

Fuoroquinolones, rifampin Inhibition of metabolic pathway

Inhibit DNA synthesis

Sulfonamides, folic acid analogues

Multidrug-resistant (MDR) strains can be defined as resistance to at least three classes of the antibiotics used in the treatment of these infections (Wróblewska, 2006). The hospital environment is the main focus for the emergence and spread of MDR bacteria. The emergence of MDR strains, usually occurs due to the selective pressure of antimicrobial therapy, i.e., inappropriate or excessive prescription of these chemicals, the frequent transmission of microorganisms and the truly large variety of mechanisms adopted by microbial cells to increase their resistance (Wróblewska, 2006). The direct relationship

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between use of antimicrobial agents and prevalence of resistant bacteria has been documented on several occasions, particularly in Intensive Care Units (ICUs) (Aarestrup, 1999).

Bacteria can adopt mechanisms conferring resistance to antibacterial drugs. Some species of bacteria are innately resistant to one or more class of antimicrobial agents and others become resistant to an antibacterial agent (Wróblewska, 2006). The organism may acquire genes encoding enzymes, such as β-lactamases, that destroy the antibacterial agent before it can have an effect; may acquire efﬂux pumps that extrude the antibacterial agent from the cell before it can reach its target; may acquire several genes for a metabolic pathway which ultimately produces altered bacterial cell walls that no longer contain the binding site of the antimicrobial agent; or may acquire mutations that limit access of antimicrobial agents to the intracellular target site (Tenover, 2006; Wróblewska, 2006).

P. aeruginosa are naturally resistant to a number of antimicrobials, such as ampicillin, amoxicillin, amoxicillin/clavulanate, cephalosporins of first and second generation, cefotaxime, ceftriaxone, nalidixic acid and trimethoprim. This intrinsic multidrug resistance occurs due to the synergy between broadly specific drug efflux pumps and the low degree of outer membrane permeability (Livermore, 2002; Pai et al., 2001; Wróblewska, 2006).

Pathogenic bacteria that express multiple mechanisms of antimicrobial resistance, are associated to high financial costs and high mortality and morbidity in humans (Tenover, 2006).

The rising prevalence of antibiotic resistance in wound bacterial pathogens represents a serious therapeutic challenge for clinicians. At the same time, the pace of development of new antibiotics has been inadequate, resulting in a shortage of novel classes of antibacterial agents to eliminate MDR pathogens. This dramatic situation has created an urgent need for developing alternative for controlling such infections, especially wound infections who do not respond to conventional antibiotic therapies. One approach is phage therapy, where the bacteriophages can be applied locally on wounds.

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1.4 Phage therapy

Phage therapy is a non-antibiotic approach to inactivate microorganisms. It involves the application of bacteriophages, as antibacterial agents to combat bacterial infections (Duckworth and Gulig, 2002; Sulakvelidze et al., 2001).

1.4.1 Discovery and history of phage therapy

In 1919, the first time in France, d'Herelle applies the phage therapy in the treatment of cholera, obtaining therapeutic success (Lorch, 1999; Sulakvelidze et al., 2001). Phage therapy was vigorously investigated and numerous studies were undertaken to assess the potential of phage therapy for the treatment of bacterial infection in humans and animals (Lorch, 1999; Skurnik et al., 2007; Summers, 2001). Early success prompted the development of multiple commercial phage preparations. For example, in 1940 Eli Lilly Company produced seven phage products for human use (Housby and Mann, 2009). These preparations were used to treat infections that cause abscesses, purulent wounds, vaginitis, acute chronic upper-respiratory tract infections and mastoid infections (Fischetti et al., 2006; Housby and Mann, 2009; Sulakvelidze et al., 2001).

However, with the development of antibiotics in the 1940s, interest in phage-based therapeutics declined in the Western world (Lorch, 1999; Sulakvelidze et al., 2001). Besides antibiotics, the most important factors that contributed to this decline was the lack of standardized testing protocols and methods of production and the beginning of World War II (Górski and Weber-Dabrowska, 2005; Lorch, 1999). Nevertheless, in Eastern Europe and the former Soviet Union, in centers such as the Eliava Institute of Bacteriophage, Microbiology and Virology in Tbilisi, Georgia and the Institute of Immunology and Experimental Therapy in Wroclaw, Poland, where access to antibiotics was limited, the development and use of phage therapy continued jointly with or in place of antibiotics

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(Lorch, 1999; Summers, 2001). It is believed that the use of phages in these countries was due to two main reasons: phage therapy was used to treat the wounds of soldiers in World War II and the treatment was cheaper (Lorch, 1999). Much of the knowledge of the application of phage therapy is due to these research centers located in these eastern countries (Lorch, 1999; Summers, 2001).

1.4.2 Pre - requisites for phage therapy

The problems related to the production of phage complicated initial study and research. Diverse stabilizers and preservatives were initially used in attempts to increase the viability of the phage therapeutics (Summers, 2001). However, because the biology of both the phage and the various stabilizers were poorly understood, many of the ingredients added in an attempt to prolong the viability of phage preparations proved to be either toxic to humans (Summers, 2001).

Another problem related to phage production was the purity grade of the preparations of these viruses. At the time, phage therapy preparations generally consisted of lysates of host bacteria that had been treated with the phage of interest (Skurnik et al., 2007). Thus, many preparations contained bacterial components (endo-and exotoxins) and products of lysis of the host that can cause some allergies or toxic effects when applied in humans (Skurnik et al., 2007). Accordingly, adverse events were often associated with the preparations, particularly in patients receiving them intravenously (Lorch, 1999).

Today, microbiologists are aware of the need for advanced purification techniques to purify phages and to ensure that they are bacterium free. The viability and titer of phages should be determined before using them therapeutically (Skurnik et al., 2007; Sulakvelidze et al., 2001; Summers, 2001). The minimum requisites needed to use the phage in phage therapy, in order to minimize possible complications are summarized in the Table 1.4.

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Table 1.4: Pre – requesites needed to use the phage in phage therapy.

Pre - requisites for phage therapy

Free of products of lysis techniques to purify phages

Well characterization phage structure, behavior in vitro and in vivo

Lytic lysogenic phages may carry genes that

encode toxins or virulence factors

Broad host range infecting members of the target species

and/or genus

Complete genome sequences know absence of any genes encoding

pathogenicity associated or potentially allergenic proteins

Sufficiently stable over storage and application

determination of viability Amenable to scale up for commercial

production

efficacy against specific bacterial and no side-effects

The phages used in phage therapy should be characterized in detail. It is necessary to sequence the genome of the phage, to identify its structure, test its behavior in vitro, and especially to prove their efficiency in vivo. Ideally, in the first place, should be tested in an animal model (Skurnik and Strauch, 2006).

For phage therapy, lytic phages should be used and the development of lysogeny must be avoided. When lysogeny is established the host becomes immune to an infection caused by the same phage or phage related (Gill and Hyman, 2010). In addition, lysogenic phages may carry genes potentially dangerous from one host to another, such as genes that encode toxins or virulence factors, which may be toxic to humans (Alisky et al., 1998; Skurnik and Strauch, 2006; Sulakvelidze et al., 2001). For these reasons, we should sequence the whole genome of the phage, which will allow us to identify genes associated with presence of lysogenic cycle, such as the integrase and repressor gene (Skurnik et al., 2007).

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1.4.3 Advantages and disadvantages of phage therapy

Advantages

There are several potential advantages of phage therapy over chemotherapy (Table 1.5).

Table 1.5: Main advantages of the phage against the antibiotics

Phages Antibiotics

Very specific Affects normal microﬂora

Low resistance High resistance

Concentrated at the local of infection

May not concentrated at the local of infection

Low costs High costs

No serious side effects Multiple side effects

One single dose Multiple doses

Phages are very specific to the target, while the antibiotics destroy pathogenic microorganisms and normal microﬂora. This affects the microbial balance in the patient, which may lead to serious secondary infections (Vinodkumar et al., 2008). The specificity of the host usually occurs at the level of strain, at the species level and rarely at the level of genus (Hagens and Loessner, 2010). The host range of phages is determined by receptors on the surface of the bacterium, allowing the binding of phage to bacteria (Skurnik and Strauch, 2006; Wróblewska, 2006). Therefore, first for an appropriate phage treatment, it will be necessary to identify the bacteria causing the infection and know which phages that infect bacterial strains. Secondly, it will be necessary to create databases with hundreds or thousands of phage preparations with different specificities (Balogh et al., 2010).

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They have limited resistance development and selecting new phages is a relatively rapid process that can frequently be accomplished in days or weeks, while the antibiotics quickly become resistant to bacteria and the development of new antibiotics may take several years (Harcombe and Bull, 2005; Skurnik and Strauch, 2006; Sulakvelidze et al., 2001).

They are safe, no serious side effects have been described, because phages or their products (amino acids and nucleic acids) do not affect eukaryotic cells (Abedon and Thomas-Abedon, 2010; Gorski et al., 2003).

The phages have the capacity to self-multiply at the site of infection, while the antibiotics do not necessarily concentrate at the site of infection (Skurnik et al., 2007). Systemic antibiotic therapy has little utility in patients with extensive wounds, because of poor penetration of the antibiotic into the wound, being the infection difficult to eliminate (Kutter et al., 2010). The reproductive ability of bacteriophage, avoids this problem. This makes phages ideal for wound treatment, in contrast to antibiotics, whose concentration decays rapidly with distance from the source and are eliminated by metabolic degradation or excretion (Brussow, 2005). Due to self-replication of the phage, the pharmacokinetics are problematic. The in vitro growth data for a phage cannot be directly applied to the in vivo situation and the in vivo data for one phage cannot be transferred to another phage. The use of phages as drugs may differ from antibiotics due to differences in the phage pharmacokinetics, which becoming the great challenge of phage therapy (Payne and Jansen, 2003). In simulations of the population and evolutionary dynamics of the phage– bacteria interactions, the phage can eliminate all of the host bacteria in the culture. However, in reality, this cannot happen. There are, at least, three reasons for this not happen. First, the phages do not infect the host bacteria when their density is below the host cell threshold (Comeau et al., 2008). Second, the host may develop resistance to the phage (Levin and Bull, 2004). Third, the bacterial population might reach stationary phase and therefore might be physiologically refractory to the phage (Levin and Bull, 2004). However, in vivo the combination of phage and the host defenses are sufficient to keep the bacterial density below lethal threshold after phage therapy. Phage therapy only needs to

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decrease the numbers of infecting bacteria to a level from which the host defenses can take care of the remaining bacteria (Levin and Bull, 2004).

Finally, phage therapy is a technology flexible, fast, cheap and efficient against MDR pathogens, since the mechanism used by phage to lyse the bacteria is different from those used by antibiotics (Matsuzaki et al., 2005; Sulakvelidze et al., 2001).

Disadvantages

One of the disadvantages of phage therapy is the possible development of bacterial resistance to the phages. In phage infection, one essential step is the attachment of the phage onto specific receptors of bacteria. By mutating in the gene that encodes a bacterial product essential for losing the phage receptor, bacteria become resistant to phages (Levin and Bull, 2004; Skurnik and Strauch, 2006). However, this resistance cannot be serious. If the receptor used by the phage is a virulence determinant, loss of the receptor would decrease the virulence of the bacterium, and then it would be easier for the host immune system to eliminate the pathogen (Levin and Bull, 2004; Skurnik and Strauch, 2006). Furthermore, even if the bacteria becoming resistant to a particular phage is easier to find a new phage that can infect the pathogen than a new antibiotic (Harcombe and Bull, 2005; Skurnik and Strauch, 2006). In addition, the rate of mutation and replication is higher in the phage, which can overcome the adaptation of bacteria (Deresinski, 2009). Finally, according to some authors, the rate of development of bacterial resistance to phage is 10 times less than the antibiotics (Carlton, 1999; Sulakvelidze et al., 2001). This rate may be much smaller if provided different phages in the same phage preparation. These cocktails of phages can be composed of two or more phages that use different receptors to infect bacteria of the same species or pathogenic bacteria more common for that particular infection (Goodridge, 2010).

The lysogenic conversion can be another problem when phages are used to infected bacteria. When lysogeny is established the phenotype of the host cell can be altered. The temperate phage (prophages) can express some genes that can result in the production of toxins and antibiotic resistance (Alisky et al., 1998; Skurnik and Strauch, 2006; Sulakvelidze

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et al., 2001). In addition, this host becomes resistant to infection by the same or similar strains of phages (Gill and Hyman, 2010).

Another drawback is the possibility of phage particles were remove by the circulatory system of the host, i.e., phages can be neutralized by antibodies. However, first, the problem can be solved if it was prepared several phage strains with different antigens (James et al., 2004). Second, Duckworth and Gulig (2002) suggest that the kinetics of phage action is much faster than the production of antibodies by the host. Therefore, this neutralization is not significant during the initial treatment of infections. The phage therapy is complete before developing specific immunity (Duckworth and Gulig, 2002).

1.4.4 Studies and applications developed in phage therapy

1.4.4.1 Eastern Europe and the former Soviet Union

In 1923 was founded the first institute of research on phage therapy, the Institute of Bacteriophage, Microbiology and Virology in Tblisi. Since 1950, the problem of antibiotic resistance was also known in the Union Soviet. Most resistant bacteria samples isolated in the Soviet Union were sent to Tblish in order to find phages corresponding to these bacteria (Lorch, 1999). Thousands of monophages and cocktail of phages (pyophage and intestiphage) for pathogenic bacteria strains, such as Staphylococcus, Streptococcus, Proteus, Pseudomonas aeruginosa and Clostrium were prepared (Kutateladze and Adamia, 2008; Lorch, 1999).

Scientist of the Eliava Institute continually renewed the cocktail pyophage and intestiphage with new phages against the most frequent and virulent strains for the prevention and treatment of wound infection and enteric bacteria, respectively (Kutateladze and Adamia, 2008). For deeper wounds, phages embedded in polymer called PhageBioderm is often used in addition to pyophage wound irrigation. PhageBioderm is a biodegradable, non-toxic polymer developed by Georgian chemists and microbiologists

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since 1995 and approved for commercial release in 2000 (Kutateladze and Adamia, 2008; Kutter et al., 2010). As a result, very broad-range and effective bacteriophage preparation were obtained and the phage sensitivity of the infections was more than 85%. These preparations were used immediately for empiric phage therapy even before the bacterial sensitivity of the phage had been tested (Kutter et al., 2010).

Research on bacteriophages was not limited to the Eliava Institute. For instance, one well-documented clinical phage therapy was carried out at the Institute for Immunology and Experimental Medicine, in Poland. While the western scientific community contributed to exchanging scientific results in English, the scientists of the Soviet Union were not included in the scientific community (Gorski et al., 2003; Lorch, 1999). However, some of these studies and their applications are being translated and provided to English-speaking scientists.

This institute, in Poland, has administrated phages against a variety of target microorganisms responsible for a number of diseases. They have a phage-bank, where they can choose one or more phages from their collections, which are active against a given bacterial isolate. Reportedly the Institute phage-bank presents over 300 specific bacteriophage strains against staphylococci, enterococci, Escherichia sp., Klebsiella sp., Salmonella sp., Shigella sp., Enterobacter sp., Proteus sp., Serratia sp., Acinetobacter sp. and Pseudomonas sp. (Kutter et al., 2010).

In the past, phage were administered orally, topically or systemically to treat a wide variety of infections, such as suppurative wound, gastroenteritis, sepsis, osteomyelitis, dermatitis, emphysemas and pneumonia (Alisky et al., 1998; Sulakvelidze et al., 2001).

Some of the clinical applications carried out in the Eastern Europe and former Soviet Union are summarized in Table 1.6.

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Table 1.6: Clinical applications of phage therapy in Eastern Europe and the Soviet Union. Adapted from Sulakvelidze et al. (2001)

Reference(s) Infection(s) Etiologic agent(s) Comments

(Babalova et al., 1968; Miliutina and Vorotyntseva, 1993; Tolkacheva et al., 1981) Bacterial dysentery and salmonellosis Shigella, Salmonella, E. coli and Proteus

The combination of phages and antibiotics was reported to be effective in treating cases where antibiotics alone were ineffective (Miliutina and Vorotyntseva, 1993). (Bogovazova et al., 1992; Cislo et al., 1987; Kochetkova et al., 1989; Sakandelidze, 1991; Weber-Dabrowska et al., 2000; Zhukov-Verezhnikov et al., 1978) Infections of skin Pseudomonas , Staphylococcus., Klebsiella spp., Proteus, E. coli and Streptococcus

31 patients having chronically infected skin ulcers were treated orally and locally with phages. The success rate was 74% (Cislo et al., 1987).

65 patients received phages and the rest received antibiotics. Phage treatment was successful in 82% of the cases, and antibiotic treatment was successful in 61% of the cases (Kochetkova et al., 1989).

(Ioseliani et al., 1980; Meladze et

al., 1982)

Lung and pleural infections

Staphylococcus, Streptococcus, E.

coli and Proteus

Phages were used to treat 223 patients and the results were compared to 117 cases where antibiotics were used. Full recovery was observed in 82% of the patients in the phage-treated group, as opposed to 64% of the patients in the antibiotic-treated group (Meladze et al., 1982). (Perepanova et al., 1995) Inﬂammatory urologic diseases Staphylococcus, E. coli, and Proteus

Adapted phages were used to treat acute and chronic urogenital inﬂammation in 46 patients. The efficacy of phage treatment was 92% (marked clinical improvements) and 84% (bacteriological clearance) (Perepanova et al., 1995). (Sakandelidze, 1991) Infectious allergoses (rhinitis, pharyngitis, dermatitis, and conjunctivitis) Staphylococcus, Streptococcus, E. coli, Proteus, enterococci, and P. aeruginosa

360 patients were treated with phages, 404 patients with antibiotics 576 patients with combination of phages and antibiotics improvement was observed in 86, 48 and 83% of the cases, respectively (Sakandelidze, 1991).

(Stroj et al., 1999) Cerebrospinal

meningitis K. pneumonia

Orally administered phages were used successfully to treat meningitis in a newborn (after antibiotic therapy failed) (Stroj et al., 1999).

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1.4.4.2 West Europe

Phage therapy research will gain momentum, while traditional antibiotic research has come to a stop in West Europe. Appropriately selected phages can easily be used to help prevent bacterial diseases in humans or animals, with potential for alternative applications and special interest for developing countries (Lorch, 1999).

The use of bacteriophage therapy requires, however, a detailed understanding of the phage-bacteria interaction and of the awareness of various novel kinetics phenomena not known in conventional drug treatments and not considered in the Eastern Europe studies (Bull et al., 2002; Levin and Bull, 2004). Kinetics theory of phage therapy predicts that the average number of phage per bacterium, that is, the multiplicity of infection (MOI), the number of phage dose applications and the timing of the phage application are important in phage therapy and are now being studied in the west countries.

In-vitro test

One critical parameter that affects phage therapy is the initial phage dose that is the multiplicity of infection (MOI). High MOI is used when the experiment requires that every cell in the culture is infected, that is, the case of phage therapy. By contrast, low MOI is used when multiple cycles of infection are required. In vitro studies allows to study what the most appropriate MOI in order to obtain an effective inactivation of the host. It has been shown in vitro conditions that the reduction of pathogenic bacteria increased with the increase of the MOI (Table 1.7). Tanji et al. (Tanji et al., 2005) showed that, in vitro, Escherichia coli concentration did not change after phage addition at a MOI of 1. When applied at a MOI of 104, the bacterial density decreased 5 logs. Andreatti Filho et. al (Andreatti Filho et al., 2007) showed that the number of viable Salmonella enteritidis decreased 4 logs at a MOI of 100. However, at a MOI of 106 the bacterial density decreased 7 logs.

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All the literature reviewed, the number of phage doses applications and the timing of the phage application were not tested in vitro.

Another critical parameter that should be tested in vitro is the host resistance developed to the phages. In most studies, the resistance of bacteria to the phage is not tested (Table 1.7). In several in vitro studies (Andreatti Filho et al., 2007; Kumari et al., 2010; Tanji et al., 2005; Watanabe et al., 2007) it was observed a gradually increased in the bacterial number during the experiments of phage therapy. The authors speculate that these results may suggest the emergence of strains resistant to the phage. However, they do not actually test experimentally the development of bacterial resistance. Nevertheless, Loc Carrillo et al. (2005) concluded that Campylobacter jejuni develop resistance to two different phages after a phage therapy experiment.

Table 1.7: In vitro study recently developed in West Europe

Host Phage MOI Result Observation Reference

Escherichia coli SP15, SP21, SP22 1 No reduction --- Resistance: Speculate (no tested) (Tanji et al., 2005) 104 Reduction of 5 logs after

8 hours of incubation A gradual increase in bacterial was observed Klebsiella pneumoniae Kpn5 0.1

Reduction of 6 logs after 3 hours of incubation A gradual increase in bacterial was observed Resistance: Speculate (no tested) (Kumari et al., 2010) Salmonella enteritidis WT45∅

100 Reduction of 4 logs after

6 hours of incubation A gradual increase in bacterial was observed Resistance: Speculate (no tested) (Andreatti Filho et al., 2007) 106 Reduction of 7 logs after

6 hours of incubation

Campylobacter

jejuni CP34 300

Reduction of 3 logs after 8 hours of incubation A gradual increase in bacterial was observed Resistance: 11% (Loc Carrillo et al., 2005) Pseudomonas aeruginosa KPP10 1

Reduction of 4 logs after 150 min of incubation A gradual increase in bacterial was observed Resistance: Speculate (no tested) (Watanabe et al., 2007) Pseudomonas aeruginosa

MPK1 10 Reduction of 5 logs after

30 min of incubation A gradual increase in bacterial was observed Resistance: not referred (Heo et al., 2009) MPK6 10 Reduction of 4 logs after

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Ex-vivo test

To understand the phage-bacteria interaction in vitro tests are not sufficient, being necessary to resort to in ex vivo tests. The ex vivo tests do not fully mimic the in vivo growth conditions, but these tests allow experimentation under identical conditions than in vivo and under more controlled conditions. This methodology combines the advantages of in vivo with the flexibility of the in vitro.

Up to my knowledge, there are a few ex vivo studies available in current literature (Atterbury et al., 2003; Goode et al., 2003). Goode et. al (Goode et al., 2003) showed that no Salmonella spp. or Campylobacter jejun were recovered, when they distributed on the surface of the chicken skin phage and their hosts at a MOI of 105. Atterbury et. al (Atterbury et al., 2003) demonstrated that Campylobacter jejuni decreased 1 log, after application of phage at a MOI of 10, on artificially contaminated chicken skin.

All the literature reviewed, the number of phage doses applications, the timing of the phage application and the host resistance developed to the phages were not tested in ex vivo.

In-vivo test

As in vivo, the appropriate MOI must be tested to effectively reduce the number of viable pathogenic bacteria and increase the survival rate of the animal model. It has been shown in vivo conditions that the survival rates increased with the increase of the MOI (Table 1.8). Huff et. al (Huff et al., 2005) demonstrated that mortality was significantly reduced from 85 to 35% at a MOI of 1, and the birds were completely protected when the challenge culture was mixed with 108 PFU/ml of bacteriophage, MOI of 10,000. Wang et. al (Wang et al., 2006) studied the dose effect of phage in rescuing mice from lethal imipenem-resistant P. aeruginosa bacteremia and showed that higher doses of the phage, MOI of 0.01-200, 100% of the animals survived. As the phage dose decreased, MOI of 0.0001 and 0.001, the animals became critically ill, showing survival rates of 0 and 20%, respectively.

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The in vivo studies have been showed that the application of a single dose appears to be sufficient to control bacterial growth, contrarily to antibiotics (Table 1.8). Biswas et. al (Biswas et al., 2002) demonstrated that a single intraperitoneal injection of 108 PFU of the phage rescued 100% of Enterococcus faecium bacteremic mice. A similar study conducted by Smith and Huggins (Smith and Huggins, 1982) showed that all mice infected by E. coli survived with a single intramuscular dose of anti-K1 phage.

Another critical parameter that can be well evaluated in vivo studies is the timing of the phage treatment. When the phage is administered too early, the phage will be released from the body before it reaches the replication threshold. When the phage is administered too late, the phage will not be effective (Table 1.8). Study by Smith and Huggins (Smith and Huggins, 1982) showed that when phages were administered in the same time that the mouse was infected with the E. coli, all mice survived. However, if the phage was administered two days after, 19 mice survived, and this number decreased to 1, when the phage was administered 7 days later. Another study done by this group showed that administration of phage 6 hours before or 18 hours after infection with E. coli, the mice developed diarrhea (Smith et al., 1987). This symptom did not happen if the phage was administered between 10 minutes before and 12 hours after infection with E. coli.

The ability of phage to reach the infection site and access the host is another critical parameter that affects the phage therapy that can be studied in vivo using animal models (Table 1.8). Several studies show that for the same type of infection, the phage can be applied through different routes and some of them are more suited than others (Jikia et al., 2005; McVay et al., 2007). McVay et. al (McVay et al., 2007) showed that the location where the phages were injected change the survival rate of mice. The mice was subjected to burn wound injury and to fatal infection with P. aeruginosa. Then, a phage cocktail was administered intramuscular (i.m.), subcutaneous (s.c.) or intraperitoneal (i.p.). The i.p. route providing the most signiﬁcant protection (87%) of the routes tested. The phages administered by the i.p. route were delivered at a higher dose, earlier and for a more sustained period of time than the phages administered by the i.m. or s.c. route. Moreover, studies have already been made in implementing the phage locally to eliminate wound

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infections instead of injecting, also showing good results. Study by Jikia et. al. (Jikia et al., 2005) demonstrated that infections in human skin caused by Staphylococcus aureus were eliminated with the application of polymers embedded in a phage solution.

Table 1.8: In vivo study recently developed in West Europe

Host Animal

model

Phage/ administration MOI Results Reference

Pseudomonas plecoglossicida

fish Cocktail phage

Orally

1 Survival rates: 80% (Park and

Nakai, 2003)

Pseudomonas aeruginosa

mice KPP10

Orally

100 Survival rates: 66.7% (Watanabe et al., 2007)

mice Cocktail phage

intraperitoneal injection 106 Survival rates: 87%

(McVay et al., 2007) intramuscular injection 106 Survival rates: 28%

subcutaneous injection 106 Survival rates: 22%

fly MPK6

Orally

1 Survival rates: 20% (Heo et al., 2009)

mice ΦA392

intraperitoneal injection

0,01 Survival rates: 100% (Wang et al., 2006) 0,0001 Survival rates: 0% Escherichia coli mice K12.K1 intramuscular injection

10 Survival rates: 94% (Smith and Huggins, 1982) Escherichia coli chickens and calves R Orally intramuscular injection

1 Survival rates: 100% (Lavigne et al., 2003)

Escherichia coli

chickens SPR02

Aerosol

1 Survival rates: 65% (Huff et al., 2005) 104 Survival rates: 100% Escherichia coli chickens F78E Orally

10 Survival rates: 25% (Oliveira et al., 2010)

Escherichia coli

mice Cocktail phage

Orally

1 Survival rates: 50% (Smith et al., 1987)

Enterococcus faecium

mice ENB6

0.1 Survival rates: 100% (Biswas et al., 2002)

Staphylococcus aureus

mice MSa

10 Survival rates: 97% (Capparelli et al., 2007)

Klebsiella pneumonia

mice KΦ1

100 Survival rates: 100% (Malik and Chhibber, 2009) Klebsiella pneumonia mice SS intranasal inhalation

100 Survival rates: 100% (Chhibber et al., 2008) Klebsiella pneumoniae mice Kpn5 intraperitoneal injection 10-200

Survival rates: 96.6% (Kumari et al., 2010) 0.1 Survival rates:53.33%

0.01 Survival rates:13.33% 0.001 Survival rates: 0%

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Clinical trails

Although the process of reintroduction of phage therapy in the West has been delayed, recently clinical cases in the West were conducted, which show the advances in clinical application of phage therapy (Table 1.9).

A recent a study, done in Switzerland, with human volunteers receiving phage T4 indicated that it is safe for oral administration (Bruttin and Brussow, 2005). No phage or phage T4-speciﬁc antibodies was detected in feces and in the serum of the human subjects. The number of E. coli in feces did not decrease and no adverse events related to phage application were reported.

In the United Kingdom, Marza and colleagues (Marza et al., 2006) reported the case of a 27 year old male with 50% surface area burns and skin grafts was applied. After several months, the patient became infected with P. aeruginosa and grafted areas broke down rapidly, despite appropriate antibiotic treatment. Therefore, treatment with phages was started. Three days after phage application, P. aeruginosa could no longer be isolated from swabs and subsequent extensive grafting was successful.

Chronic otitis is a very common disease and very difficult to treat. Here, P. aeruginosa are often largely organized into biofilms and relatively protected from both antibiotics and immune cells, being particularly hard to eradicate. The Biocontrol scientists conducted a successful trial of phage against Pseudomonas dog ear infections (Wright et al., 2009). The results of that trial were necessary to obtain regulatory approval for a phase I/II in human trial. In humans infected with Psedudomonas sp., they applied a single dose of a phage cocktail with six different phages. The controlled clinical trial of a therapeutic bacteriophage preparation showed efficacy and safety. The company is now pursuing a phase III trial in the near future.

Another phase I clinical study was performed to treat patients with venous leg ulcers (Rhoads et al., 2009). The cocktail phage, developed by Intralytix, contained eight individual phages (five were lytic for P. aeruginosa, two for S. aureus and one for E. coli).

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Forty two patients with full thickness venous leg ulcers of over 30 days duration were included in the study. Patients received 50 ml of either diluted phage preparation or of sterile saline. Results of the study revealed no significant differences was determined between the test and control groups for frequency of adverse events, rate of healing, or frequency of healing. Efficacy of the preparation will need to be evaluated in a phase II. Some pre-clinical studies are already in study to inactive different bacteria, namely methicillin-resistant S. aureus strains (Table 1.9).

Table 1.9: Clinical trials of phage therapy in West Europe Clinical infection/

bacterial agent Product Stage development of References

Healthy human Orally phage Pre-clinical (Bruttin and Brussow,

2005)

P. aeruginosa

Burns infections

Discs soaked with phage solution

Phase I/ II (Marza et al., 2006)

P. aeruginosa

Ears infections

Cocktail of phage Phase I/ II (Wright et al., 2009)

Venous leg ulcer infections

Cocktail of phage Phase I (Rhoads et al., 2009)

Mycoplasma mycoides Vaccines (orally phage)

Phase I/ II (March et al., 2006)

E. coli, Staphylococcus

sp., Streptococcus sp.,

Pseudomonas sp.

Phage for clinical trials

Pre-clinical (BiophagePharma)(Can

ada)

S. aureus Phage for clinical trials

Pre-clinical (Gangagen) (India and

USA) Methicillin-resistant S.

aureus

Pre-clinical (Novolytics) (United

Kingdom) Methicillin-resistant S.

aureus, C . difficile, E. coli, K. pneumoniae

and P. aeruginosa

Phage products Pre-clinical and phase I (PhicoTherapeutics) (United Kingdom)

Pseudomonas sp Phage for clinical trials

Pre-clinical (PhageBiotech) (Israel)

Methicillin-resistant S.

aureus and P. aeruginosa

Pre-clinical (SpecialPhageHoldings)

(Australia)

S. aureus.

Wound, systemic and respiratory infections

Pre-clinical (Viridax) (USA)

In Portugal, there are two major companies also involved in the investigation of phage products, the Technophage SA in Lisbon and the Innophage in Oporto.

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1.4.5 Phage therapy studies against Pseudomonas aeruginosa

Different phages have been tested to inactivate a variety of P. aeruginosa strains (Table 1.10) and in general, all these phage-bacteria interaction studies reveal that phages are capable of decreasing the number of viable bacteria, increasing the survival rate of the hosts (in vivo studies). Most of these studies did not evaluate the development of resistance by the bacteria (Table 1.10). The resistance development was only studied in two of the 28 studies considered in this revision and the results are very discrepant. Further studies are necessary to evaluate the importance of resistance development during phage therapy.

Table 1.10: Use of bacteriophages to control Pseudomonas aeruginosa

References Infections Tested Comments Resistance Phage(s)

In vivo

(Merabishvili et al., 2009)

burn wound Humans Stable, sterile and no

cytotoxic

- Cocktail-BFC1

(Rhoads et al., 2009) venous leg ulcers Humans Efficacy and safety - - (Wright et al., 2009) chronic otitis Humans Efficacy and safety - Biophage-PA (Marza et al., 2006) burn wound Humans No recovered P.

aeruginosa

- -

(Marza et al., 2006) chronic otitis Dog No recovered P.

aeruginosa

- -

(Hawkins et al., 2010)

otitis Dog Redution: 56% - Cocktail

(Heo et al., 2009) systemic infection

Fly Survival: 20% - MPK6

(Soothill, 1994) burn wound Guinea-pig Control P.

aeruginosa

- -

(Velásquez, 2011) thermal injuries Mice Survival:100%-28% - Pa02

(Heo et al., 2009) peritonitis sepsis Mice Survival:100%-40% - MPK6, MPK1

(McVay et al., 2007) burn wound Mice Survival:87%-22% Resistance: 0% Cocktail (Vinodkumar et al.,

2008)

septicemia Mice Survival:100% - CSV-31.

(Wang et al., 2006) bacteremia Mice Survival:100% - ΦA392

(Watanabe et al., 2007)

gut-derived Sepsis

Phage therapy to inactivate multidrug-resistant P. aeruginosa

Anabela Carvalho

Vieira

Phage therapy to inactivate multidrug-resistant

P. aeruginosa

Terapia fágica para inactivar P. aeruginosa

multi-resistente

Anabela Carvalho

Vieira

Phage therapy to inactivate multidrug-resistant

P. aeruginosa

Terapia fágica para inactivar P. aeruginosa

multi-resistente

Table of Contents

1. Introduction

1.1.

Bacteriophages

1.2.

Human skin flora and wound infection

Infections by Pseudomonas aeruginosa

1.3

Bacterial resistance to antibiotics

1.4

Phage therapy

Advantages

Disadvantages

In-vitro test

Ex-vivo test

In-vivo test

Clinical trails